Methods and compositions for detecting anti-hepatitis E virus activity

ABSTRACT

The present invention provides antigenic peptides and polypeptides of hepatitis E virus. Also provided are mixtures of conjugated and unconjugated peptides of the present invention. Methods of detecting hepatitis E viral infection in a subject using the peptides and peptide mixtures of the present invention are also contemplated.

This application is a division of application Ser. No. 07/965,667, filedOct. 21, 1992, which status is pending.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The invention pertains to methods and compositions for detectinganti-hepatitis E virus activity in a subject. The compositions includeantigenic peptides of hepatitis E virus and mixtures of antigenicpeptides of hepatitis E virus. The methods include serologic diagnosisof hepatitis E viral infection using the peptides and peptide mixturesof this invention.

2. Background Art

Hepatitis E virus (HEV) is a recently discovered agent of entericallytransmitted non-A, non-B hepatitis (ET-NANB). The disease remains aserious problem in many developing countries. Unlike other agents ofviral hepatitis, HEV infection is often associated with high mortalityrates in infected pregnant women.

The first reported outbreak of ET-NANB hepatitis occurred in New Delhi,India in 1955. However, only after serologic tests for IgManti-hepatitis A virus became available to exclude hepatitis A virus asthe cause, was this very large outbreak recognized as ET-NANB hepatitis.Since that time epidemics of ET-NANB infection have been documented inmany countries.

Until recently, the diagnosis of ET-NANB hepatitis outbreaks could onlybe based upon the absence of serologic markers of hepatitis A virus(HAV) and hepatitis B virus (HBV). Subsequently, specific tests for thedetection of the ET-NANB hepatitis were based upon immune electronmicroscopy (IEM), in which a small volume of a stool suspension fromacutely infected individuals is incubated with acute- orconvalescent-phase sera and examined by electron microscopy (Bradley etal. PNAS USA 1987;84:6277-6281, 1987). IEM, thus identified 27-32 nmvirus-like particles using acute and convalescent phase sera as thesource of antibody. However, since most clinical specimens do notcontain sufficient virus-like particles to visualize using IEM, thismethod is not useful for clinical or epidemiological analysis.

More recently, Reyes et al. (Science 247:1335-1339, 1990) successfullyisolated and sequenced a partial cDNA clone from HEV. The HEV genome hassubsequently been characterized as an RNA positive strand virus with anorganization similar to Caliciviruses. Three open reading frames (ORF)have been identified (Tam et al. Virology, 185:120-131, 1991). Twotype-common HEV epitopes were identified in proteins encoded by ORF2 andORF3 (Reyes et al. Gastroenterologia Japonica 26 (suppl.3): 142-147,1991b; Ichikawa et al. Immunol. 35:535-543, 1991). Both are localized atthe C-terminus of their respective proteins. These epitopes wereexpressed as hybrid proteins with beta-galactosidase orglutathione-S-transferase and were recognized or an enzyme immunoassay?]by antibodies from acute- and convalescent-phase sera obtained fromexperimentally infected cynomologus macaques (Reyes et al., in "Viralhepatitis C,D,E", T. Shikata, R.H. Purcell, T. Uchida (Eds.) ElsevierScience Publishers, NY, pp.237-245, 1991a) or humans (Goldsmith et al.,Lancet 339:328-331, 1992). These hybrid proteins have the disadvantagethat the chimeric part of protein can negatively influence folding.Furthermore, individuals may have antibodies expressed to thesesequences.

ORF2 has been suggested to be responsible for the expression of the HEVstructural protein(s) (Tam et al., 1991). In addition, the recombinantpolypeptide containing the C-terminal half of the protein has been shownto be an important diagnostic reagent for the detection of anti-HEVactivity in patients infected with HEV.

Reyes et al. (l991a) demonstrated that a short fragment of theC-terminal region of the protein encoded by ORF3, obtained by expressionof DNA derived from the HEV genome of the Burma strain did not reactwith sera from cynomologous macaques infected with the Mexico strain ofHEV. Conversely, expressed recombinant protein derived from the Mexicostrain did not react with sera from macaques infected with the Burmastrain of HEV (Yarbough et al. J. Virol. 65:5790-5797, 1991). Sequencecomparison of the two strains at the C-terminal region of ORF3 revealeda 78% homology (Yarbough et al., 1991). Furthermore, there appear to betype-common viral epitopes that are shared by divergent geographicisolates from Asia and North America (Yarbough et al. 1991; Goldsmith etal., 1992).

Thus, because of the lack of sensitivity and difficulty of performingthe previously available tests, there exists a need for a rapid, simpleand highly sensitive diagnostic test for HEV infection.

The present invention meets these needs by providing synthetic peptidesand their use in a diagnostic test for the detection of antibodies tothe hepatitis E virus. The present invention provides for theapplication of synthetic peptides in an immunodiagnostic assay for thedetection of antibodies to HEV (anti-HEV).

SUMMARY OF THE INVENTION

The present invention provides antigenic peptides of HEV. For examplethe peptides of the present invention can consist of the amino acidscontained in the amino acid sequences defined in the Sequence Listing bySEQ ID NOS: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or 13. The peptides ofthe present invention can be unconjugated, or they can be conjugated toa carrier molecule that facilitates placement of the peptide on thesolid phase. Also provided is a composition comprising at least fourdifferent peptides, wherein the peptides are defined in the SequenceListing by SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:7, SEQ ID NO:8 and SEQ IDNO:9.

This invention further provides a method of using the composition ofdifferent peptides for detecting hepatitis E viral infection in asubject by contacting an antibody-containing sample from the subjectwith a detectable amount of the peptides and detecting the reaction ofthe peptides and antibody specifically reactive therewith, the reactionindicating the presence of hepatitis E infection.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1(A) and 1(B) show hydropathy plots of the proteins encoded by HEVORF2 (A) and ORF3 (B), and the localization of sequences selected forthe synthesis of peptides;

FIG. 2 shows the frequency distribution of anti-HEV activity; and

FIG. 3 shows the results of a neutralization test using sera whichinitially tested positive and yielded optical density values from 0.1 togreater than 2.5.

DETAILED DESCRIPTION OF THE INVENTION

Antigen

The present invention provides antigenic polypeptide fragments orpeptides of HEV. The peptides generally exist in a purified form. Asused herein, "purified" means the peptide is essentially free ofnaturally occurring contaminants. The purified antigenic HEV peptides orpolypeptides of the present invention are also referred to herein as"the antigen" or "the HEV antigen" and are designated interchangeably byeither peptide number or SEQ ID NO (Tables 1 and 2).

The peptides of the present invention can consist essentially of theamino acids contained in the amino acid sequences defined in theSequence Listing by SEQ ID NOS: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or13. Thus, the peptides of the invention have the same general sequenceas found in the Sequence Listing. The peptides of the present inventioncan be unconjugated, or they can be conjugated to a carrier moleculethat facilitates placement of the peptide on the solid phase. A carrierprotein is one to which synthetic peptides can be conjugated and whichwill not react with antibodies in human serum. An example of such acarrier is bovine serum albumin (BSA).

Once the amino acid sequence of the antigen is provided, it is possibleto synthesize, using the methods taught herein and standard peptidesynthesis techniques, peptide fragments chosen to be homologous toimmunoreactive regions of the antigen and to modify these fragments byinclusion, deletion or modification of particular amino acid residues inthe derived sequences. Thus, synthesis or purification of an extremelylarge number of peptides derived from the antigen is possible.

Alternatively, an antigenic peptide can be isolated from the wholeantigen by chemical or mechanical disruption. The purified peptides thusobtained can be tested to determine their antigenicity and specificityby the methods taught herein. An immunoreactive peptide is defined as anamino acid sequence of at least about 5 consecutive amino acids derivedfrom the antigen amino acid sequence.

The peptide/polypeptide fragments of the present invention can also berecombinant proteins obtained by cloning nucleic acids encoding thepolypeptide in an expression system capable of producing the antigenicpeptide/polypeptide.

The amino acid sequences of the present peptides/polypeptides cancontain an immunoreactive portion of HEV antigen attached to sequencesdesigned to provide for some additional property, such as solubility astaught herein. The amino acid sequences of an HEV antigen can includesequences in which one or more amino acids have been substituted withanother amino acid to provide for some additional property, such as toremove/add amino acids capable of disulfide bonding to increase thereactivity of an epitope by providing a more rigid secondary structure,to increase its bio-longevity or to alter its cytotoxicity or to preventinfection. In any case, the peptide must posses immunoreactivity andimmunogenicity.

Peptide mixtures

The present invention also provides mixtures (compositions) of thepeptides provided herein as illustrated, for example, by mixtures 5, 7and 9 described in the Example 2. In addition to the individualpeptides, the mixtures of the present invention can also be referred toherein as "the antigen" or "the HEV antigen." As with the individualpeptides, the mixtures of this invention can comprise conjugatedpeptides, unconjugated peptides or both. Furthermore, the conjugatedpeptides of the invention can be amounts of an individual peptideconjugated to a carrier (for example, mixture 7) or amounts of differentpeptides conjugated to a single carrier (for example, mixture 9). Themixtures, as well as the individual peptides, can be attached or boundto a substrate (solid phase).

Specifically, the present invention provides a composition comprising atleast four different peptides, wherein the peptides have a sequencewhich consists essentially of the amino acids contained in the aminoacid sequences defined in the Sequence Listing by SEQ ID NO:1, SEQ IDNO:2, SEQ ID NO:8 and SEQ ID NO:9. The peptides of this mixture can beconjugated, individually or as a mixture, to a carrier and can then bebound to a substrate. When this composition further comprises thepeptide consisting essentially of the amino acids contained in the aminoacid sequence defined in the Sequence Listing by SEQ ID NO:7, mixture 7is provided.

Also provided is a composition comprising at least eight peptides,wherein the peptides have a sequence which consists essentially of theamino acids contained in the amino acid sequences defined in theSequence Listing by SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:6,SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10 and SEQ ID NO:11. The peptides ofthis mixture are conjugated, individually or as a mixture, to a carrierand can then be bound to a substrate.

Other mixtures of peptides of the present invention can, for exampleinclude the following:

Peptide 2 and one or more of peptides 3, 5, 6, 11, 12, 13, 22, 23, 28,29, 33 and 40; Peptide 3 and one or more of peptides 2, 5, 6, 11, 12,13, 22, 23, 28, 29, 33 and 40; Peptide 5 and one or more of peptides 2,3, 6, 11, 12, 13, 22, 23, 28, 29, 33 and 40; Peptide 6 and one or moreof peptides 2, 3, 5, 11, 12, 13, 22, 23, 28, 29, 33 and 40; Peptide 11and one or more of peptides 2, 3, 5, 6, 12, 13, 22, 23, 28, 29, 33 and40; Peptide 12 and one or more of peptides 2, 3, 5, 6, 11, 13, 22, 23,28, 29, 33 and 40; Peptide 13 and one or more of peptides 2, 3, 5, 6,11, 12, 22, 23, 28, 29, 33 and 40; Peptide 22 and one or more ofpeptides 2, 3, 5, 6, 11, 12, 13, 23, 28, 29, 33 and 40; Peptide 23 andone or more of peptides 2, 3, 5, 6, 11, 12, 13, 22, 28, 29, 33 and 40;Peptide 28 and one or more of peptides 2, 3, 5, 6, 11, 12, 13, 22, 23,29, 33 and 40; Peptide 29 and one or more of peptides 2, 3, 5, 6, 11,12, 13, 22, 23, 28, 33 and 40; Peptide 33 and one or more of peptides 2,3, 5, 6, 11, 12, 13, 22, 23, 28, 29 and 40; and Peptide 40 and one ormore of peptides 2, 3, 5, 6, 11, 12, 13, 22, 23, 28, 29 and 33. Thesepeptides are shown individually in Tables 1 and 2.

Determining Antigenicity/Immunoreactivity

A method of selecting alternative peptides having immunoreactivity withan antibody reactive with the peptides of this invention is alsoprovided. For example, such a method for determining the minimalsequence for immunoreactivity of a peptide having immunoreactivity withan antibody reactive with HEV includes the following steps: (a)modifying a peptide of the present invention; (b) contacting themodified peptide with a confirmed HEV positive serum sample from asubject; and (c) detecting the reaction of the modified peptide andanti-HEV antibody, the reaction indicating that the modified peptide hasimmunoreactivity with HEV. An example of this method, which can beapplied to the other peptides of the present invention, is illustratedin Example 1. Any of the peptides of the invention can likewise bemodified.

Determining Immunogenicity

The purified peptide/polypeptide fragments thus obtained can be testedto determine their immunogenicity and specificity. Briefly, variousconcentrations of a putative immunogenically specific peptide areprepared and administered to an animal and the immunological response(i.e., the production of antibodies) of an animal to each concentrationis determined. The amounts of antigen administered depend on thesubject, e.g. a human or other susceptible animal, the condition of thesubject, the size of the subject, etc. Thereafter aninfection-susceptible animal so inoculated with the antigen can beexposed to the virus to test the potential vaccine effect of thespecific immunogenic peptide. The specificity of a putative immunogenicpeptide can be ascertained by testing sera or other fluid from theinoculated animal for cross reactivity with other closely relatedviruses. Alternatively, the immunogenicity can be tested in an in vitromethod using serum from the immunized animal to attempt to neutralizeinfectious virus, which can then be added to cell culture to determineif the peptide elicited neutralizing antibodies.

Recombinant Mosaic Proteins

Because the present invention provides the amino acid sequences ofantigenic peptides and their nucleic acid coding sequences in the HEVgenome (Yarborough et al., 1991), a recombinant mosaic protein can beproduced comprising a plurality of the peptides of the presentinvention.

The protein can include the epitopes of peptides 5, 6, 22, 23, 33 and40, among others, and can also include additional amino acids that donot substantially affect the antigenicity of the protein. This mosaicprotein is highly sensitive and specific because of the absence ofextraneous amino acids that can interfere with the presentation of theepitopes. It is contemplated that the mosaic proteins of this inventioncan be used, as described herein, for diagnostic tests and vaccines. Thecurrently preferred method of expressing the mosaic protein is by meansof vector-host expression systems.

Vectors and Hosts

A vector comprising the nucleic acids of the present invention is alsoprovided. The vectors of the invention can be in a host capable ofexpressing the antigen. Making and using such vectors and hosts, usingthe teachings of the present invention, is within the level of skill ofthose in the art.

There are numerous E. coli expression vectors known to one of ordinaryskill in the art useful for the expression of the antigen. Othermicrobial hosts suitable for use include bacilli, such as Bacillussubtilus, and other enterobacteriaceae, such as Salmonella, Serratia,and various Pseudomonas species. In these prokaryotic hosts one can alsomake expression vectors, which will typically contain expression controlsequences compatible with the host cell (e.g., an origin ofreplication). In addition, any number of a variety of well-knownpromoters will be present, such as the lactose promoter system, atryptophan (Trp) promoter system, a beta-lactamase promoter system, or apromoter system from phage lambda. The promoters will typically controlexpression, optionally with an operator sequence, and have ribosomebinding site sequences, for example, for initiating and completingtranscription and translation. If necessary an amino terminal methioninecan be provided by insertion of a Met codon 5' and in-frame with theantigen. Also, the carboxy-terminal extension of the antigen can beremoved using standard oligonucleotide mutagenesis procedures.

Additionally, yeast expression can be used. There are several advantagesto yeast expression systems. First, evidence exists that proteinsproduced in a yeast secretion systems exhibit correct disulfide pairing.Second, post-translational glycosylation is efficiently carried out byyeast secretory systems. The Saccharomyces cerevisiaepre-pro-alpha-factor leader region (encoded by the MFα-1 gene) isroutinely used to direct protein secretion from yeast. The leader regionof pre-pro-alpha-factor contains a signal peptide and a pro-segmentwhich includes a recognition sequence for a yeast protease encoded bythe KEX2 gene: this enzyme cleaves the precursor protein on the carboxylside of a Lys-Arg dipeptide cleavage-signal sequence. The antigen codingsequence can be fused in-frame to the pre-pro-alpha-factor leaderregion. This construct is then put under the control of a strongtranscription promoter, such as the alcohol dehydrogenase I promoter ora glycolytic promoter. The antigen coding sequence is followed by atranslation termination codon which is followed by transcriptiontermination signals. Alternatively, the antigen coding sequences can befused to a second protein coding sequence, such as Sj26 orβ-galactosidase, used to facilitate purification of the fusion proteinby affinity chromatography. The insertion of protease cleavage sites toseparate the components of the fusion protein is applicable toconstructs used for expression in yeast.

Mammalian cells permit the expression of proteins in an environment thatfavors important post-translational modifications such as folding andcysteine pairing, addition of complex carbohydrate structures, andsecretion of active protein. Vectors useful for the expression ofantigen in mammalian cells are characterized by insertion of the antigencoding sequence between a strong viral promoter and a polyadenylationsignal. The vectors can contain genes conferring either Gentamicin ormethotrexate resistance for use as selectable markers. The antigen andimmunoreactive peptide coding sequence can be introduced into a Chinesehamster ovary cell line using a methotrexate resistance-encoding vector.Presence of the vector DNA in transformed cells can be confirmed bySouthern analysis and production of an RNA corresponding to the antigencoding sequence can be confirmed by Northern analysis. A number of othersuitable host cell lines capable of secreting intact human proteins havebeen developed in the art, and include the CHO cell lines, HeLa cells,myeloma cell lines, Jurkat cells, etc. Expression vectors for thesecells can include expression control sequences, such as an origin ofreplication, a promoter, an enhancer, and necessary informationprocessing sites, such as ribosome binding sites, RNA splice sites,polyadenylation sites, and transcriptional terminator sequences.Preferred expression control sequences are promoters derived fromimmunoglobulin genes, SV40, Adenovirus, Bovine Papilloma Virus, etc. Thevectors containing the DNA segments of interest can be transferred intothe host cell by well-known methods, which vary depending on the type ofcellular host. For example, calcium chloride transfection is commonlyutilized for prokaryotic cells, whereas calcium phosphate treatment orelectroporation can be used for other cellular hosts.

Alternative vectors for the expression of antigen in mammalian cells,similar to those developed for the expression of human gamma-interferon,tissue plasminogen activator, clotting Factor VIII, hepatitis B virussurface antigen, protease Nexinl, and eosinophil major basic protein,can be employed. Further, the vector can include CMV promoter sequencesand a polyadenylation signal available for expression of inserted DNAsin mammalian cells (such as COS7).

The DNA sequences can be expressed in hosts after the sequences havebeen operably linked to, i.e., positioned to ensure the functioning of,an expression control sequence. These expression vectors are typicallyreplicable in the host organisms either as episomes or as an integralpart of the host chromosomal DNA. Commonly, expression vectors cancontain selection markers, e.g., tetracycline resistance or hygromycinresistance, to permit detection and/or selection of those cellstransformed with the desired DNA sequences (see, e.g., U.S. Pat. No.4,704,362).

Polynucleotides encoding a variant peptide/polypeptide can includesequences that facilitate transcription (expression sequences) andtranslation of the coding sequences such that the encodedpeptide/polypeptide product is produced. Construction of suchpolynucleotides is well known in the art. For example, suchpolynucleotides can include a promoter, a transcription termination site(polyadenylation site in eukaryotic expression hosts), a ribosomebinding site, and, optionally, an enhancer for use in eukaryoticexpression hosts, and, optionally, sequences necessary for replicationof a vector.

Purified Antibodies

A purified monoclonal antibody specifically reactive with the antigen isalso within the scope of the invention. The antibodies can bespecifically reactive with a unique epitope of the antigen or they canalso react with epitopes of other organisms. The term "reactive" meanscapable of binding or otherwise associating nonrandomly with an antigen."Specifically reactive" as used herein describes an antibody or otherligand that does not cross react substantially with any antigen otherthan the one specified, in this case, the HEV antigen. Antibodies can bemade as described in Harlow and Lane (Antibodies; A Laboratory Manual,Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 1988). Briefly,purified antigen can be injected into an animal in an amount and inintervals sufficient to elicit an immune response. Antibodies can eitherbe purified directly, or spleen cells can be obtained from the animal.The cells are then fused with an immortal cell line and screened forantibody secretion. The antibodies can be used to screen DNA clonelibraries for cells secreting the antigen. Those positive clones canthen be sequenced as described in the Examples or by other methods (see,for example, Kelly et al., Bio/Technology 10:163-167, 1992 andBebbington et al., Bio/Technology 10:169-175, 1992). Purified nonhuman,preferably mammalian, polyclonal antibodies reactive with the HEVantigenic peptides provided herein are also contemplated. The polyclonalantibody can also be obtained by the standard immunization andpurification protocols (Harlow and Lane, 1988).

The antibody can be bound to a substrate or labeled with a detectablemoiety or both bound and labeled. The detectable moieties contemplatedwith the composition of the present invention are those listed below inthe description of the diagnostic methods, including fluorescent,enzymatic and radioactive markers.

Serological Detection (Diagnosis) Methods

Detecting Antibody with Antigen

The present invention provides a method of detecting HEV infection in asubject, comprising the steps of contacting an antibody-containingsample from the subject with a detectable amount of an HEV antigenicpeptide or peptide mixtures of the present invention, under suitablereaction conditions, and detecting the reaction of the peptide and theantibody specifically reactive therewith, the reaction indicating thepresence of HEV or previous infection with HEV.

Detectable amounts of the present peptides can be determined empiricallyonce their sequence and antigenicity are provided. The concentration ofan individual peptide in a mixture can also be determined empirically.Peptides with higher conjugation efficiency will be added to themixtures in lower concentrations than less efficiently conjugatedpeptides. Examples of detectable concentrations of peptides combined ina mixture are provided in mixtures 5, 7, 9 described in Example 2. Forinstance mixtures 7 and 9 are compositions in which, prior to theconjugation step, the preferred concentration of the peptide defined bySEQ ID NO:1 is about 8 micrograms per milliliter, the concentration ofthe peptide defined by SEQ ID NO:2 is about 14 micrograms permilliliter, the concentration of the peptide defined by SEQ ID NO:7 isabout 4 micrograms per milliliter, the concentration of the peptidedefined by SEQ ID NO:8 is about 4 micrograms per milliliter, and theconcentration of the peptide defined by SEQ ID NO:9 is about 20micrograms per milliliter.

It is also understood that ranges of concentration ratios including theabove values can be determined that are also antigenically effective anddetectable. For example, the concentration ratio of the peptides definedby SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:7, SEQ ID NO:8 and SEQ ID NO:9can be about 8:14:4:4:20. The determination of such effective ratiosshould also take into consideration the ratio of peptide to carrier astaught in Example 2.

Detecting Antigen with Antibody/Ligand

One example of the method of detecting HEV possessing the antigen isperformed by contacting a fluid or tissue sample from the subject withan amount of a purified antibody specifically reactive with the antigen,and detecting the reaction of the ligand with the antigen. It iscontemplated that the antigen will be on an intact HEV virion, onHEV-infected cells expressing the antigen, or will be fragments of theantigen. As contemplated herein, the antibody includes any ligand whichbinds the antigen, for example, an intact antibody, a fragment of anantibody or another reagent that has reactivity with the antigen. Thefluid sample of this method can comprise any body fluid which wouldcontain the antigen or a cell containing the antigen, such ascerebrospinal fluid, blood, bile, plasma, serum, saliva and urine. Otherpossible examples of body fluids include sputum, mucus and the like.

ELISA

Enzyme immunoassays such as immunofluorescence assays (IFA), enzymelinked immunosorbent assays (ELISA) and immunoblotting can be readilyadapted to accomplish the detection of the HEV antibodies. An ELISAmethod effective for the detection of the antibodies can, for example,be as follows: (1) bind the antigen to a substrate; (2) contact thebound antigen with a fluid or tissue sample containing the antibody; (3)contact the above with a secondary antibody bound to a detectable moietywhich is reactive with the bound antibody (e.g., horseradish peroxidaseenzyme or alkaline phosphatase enzyme); (4) contact the above with thesubstrate for the enzyme; (5) contact the above with a color reagent;(6) observe color change.

Competitive Inhibition Assay

Another immunologic technique that can be useful in the detection of HEVor previous HEV infection utilizes monoclonal antibodies (MAbs) fordetection of antibodies specifically reactive with HEV antigen. Briefly,sera from the subject is reacted with the antigen bound to a substrate(e.g. an ELISA 96-well plate). Excess sera is thoroughly washed away. Alabeled (enzyme-linked, fluorescent, radioactive, etc.) monoclonalantibody is then reacted with the previously reacted antigen-serumantibody complex. The amount of inhibition of monoclonal antibodybinding is measured relative to a control (no patient serum antibody).The degree of monoclonal antibody inhibition is a very specific test fora particular variety or strain since it is based on monoclonal antibodybinding specificity. MAbs can also be used for detection directly incells by IFA.

Micro-Agglutination Assay

A micro-agglutination test can also be used to detect the presence ofHEV in a subject. Briefly, latex beads, red blood cells or otheragglutinable particles are coated with the antigen and mixed with asample from the subject, such that antibodies in the tissue or bodyfluids that are specifically reactive with the antigen crosslink withthe antigen, causing agglutination. The agglutinated antigen-antibodycomplexes form a precipitate, visible with the naked eye or byspectrophotometer. In a modification of the above test, antibodiesspecifically reactive with the antigen can be bound to the beads andantigen in the tissue or body fluid thereby detected.

Sandwich Assay/Flow Cytometry/Immunoprecipitation

In addition, as in a typical sandwich assay, the antibody can be boundto a substrate and reacted with the antigen. Thereafter, a secondarylabeled antibody is bound to epitopes not recognized by the firstantibody and the secondary antibody is detected. Since the presentinvention provides HEV antigen for the detection of HEV or previous HEVinfection other serological methods such as flow cytometry andimmunoprecipitation can also be used as detection methods.

In the diagnostic methods taught herein, the antigen can be bound to asubstrate and contacted by a fluid sample such as blood, serum, urine orsaliva. This sample can be taken directly from the patient or in apartially purified form. In this manner, antibodies specific for theantigen (the primary antibody) will specifically react with the boundantigen. Thereafter, a secondary antibody bound to, or labeled with, adetectable moiety can be added to enhance the detection of the primaryantibody. Generally, the secondary antibody or other ligand which isreactive, either specifically with a different epitope of the antigen ornonspecifically with the ligand or reacted antibody, will be selectedfor its ability to react with multiple sites on the primary antibody.Thus, for example, several molecules of the secondary antibody can reactwith each primary antibody, making the primary antibody more detectable.

Detectable Moieties

The detectable moiety will allow visual detection of a precipitate or acolor change, visual detection by microscopy, or automated detection byspectrometry, radiometric measurement or the like. Examples ofdetectable moieties include fluorescein and rhodamine (for fluorescencemicroscopy), horseradish peroxidase (for either light or electronmicroscopy and biochemical detection), biotin-streptavidin (for light orelectron microscopy) and alkaline phosphatase (for biochemical detectionby color change).

Detecting Disease

Because the purified HEV antigen provided herein is associated withdisease, the present invention also provides a method of detecting HEVassociated disease syndrome in a subject. The method can be accomplishedaccording to the methods set forth above for the detection of HEVantigen and antibodies specifically reactive therewith. The presence ofthe HEV antigen or anti-HEV antibodies indicates the presence of diseasesyndrome in the subject.

The present invention also provides a method for diagnosing the acutephase of hepatitis E infection in a subject by contacting anantibody-containing sample from the subject with a detectable amount ofa peptide or mixture having a relatively rapidly decaying immuneresponse and detecting the reaction of the peptides and antibodyspecifically reactive therewith, the reaction indicating acute hepatitisE infection.

Vaccines

The antigen of this invention can be used in the construction of avaccine comprising an immunogenic amount of the antigen and apharmaceutically acceptable carrier. The vaccine can be a peptide of thepresent invention or the peptide bound to a carrier or a mixture ofbound or unbound peptides or an epitope specific to the antigen or itcan be potentially reactive with antibodies to other infectious agents.The vaccine can then be used in a method of preventing HEV infection.

Immunogenic amounts of the antigen can be determined using standardprocedures. Briefly, various concentrations of a putative specificimmunoreactive peptides or polypeptides are prepared, administered to ananimal and the immunological response (e.g., the production ofantibodies or cell-mediated response) of an animal to each concentrationis determined.

The pharmaceutically acceptable carrier in the vaccine of the instantinvention can comprise saline or other suitable carriers (Arnon, R.(Ed.) Synthetic Vaccines I:83-92, CRC Press, Inc., Boca Raton, Fl.,1987). An adjuvant can also be a part of the carrier of the vaccine, inwhich case it can be selected by standard criteria based on the antigenused, the mode of administration and the subject (Arnon, R. (Ed.),1987). Methods of administration can be by oral or sublingual means, orby injection, depending on the particular vaccine used and the subjectto whom it is administered.

It can be appreciated from the above that the vaccine can be used as aprophylactic or a therapeutic. Thus, the invention provides methods ofpreventing or treating an HEV infection and the associated disease byadministering the vaccine to a subject.

Antibody-Detecting Kit

The diagnostic kit of the present invention can be used to detect thepresence of a primary antibody specifically reactive with HEV or anantigenic peptide thereof. The kit can include the HEV antigen of thepresent invention bound to a substrate, a secondary antibody reactivewith the antibody specifically reactive with the HEV antigen and areagent for detecting a reaction of the secondary antibody with theprimary antibody. Such a kit can be an ELISA kit and can comprise thesubstrate, antigen, primary and secondary antibodies when appropriate,and any other necessary reagents such as detectable moieties, enzymesubstrates and color reagents as described above. The diagnostic kitcan, alternatively, be an immunoblot kit generally comprising thecomponents and reagents described herein.

Antigen-Detecting Kit

The present invention provides a kit for the diagnosis of infection bystrains of HEV possessing the HEV antigen. Particularly, the kit candetect the presence of HEV antigen or an immunoreactive peptide thereofspecifically reactive with an antibody. The kit can include an antibodybound to a substrate, a secondary antibody reactive with the antigen anda reagent for detecting a reaction of the secondary antibody with theantigen. Such a kit can be an ELISA kit and can comprise the substrate,primary and secondary antibodies when appropriate, and any othernecessary reagents such as detectable moieties, enzyme substrates andcolor reagents as described above. The diagnostic kit can,alternatively, be an immunoblot kit generally comprising the componentsand reagents described herein.

The particular reagents and other components included in the diagnostickits of the present invention can be selected from those available inthe art in accord with the specific diagnostic method practiced in thekit. Such kits can be used to detect the antigen in tissue and fluidsamples from a subject.

The following examples are intended to illustrate, but not limit, theinvention. While they are typical of those that might be used, otherprocedures known to those skilled in the art may be alternativelyemployed.

EXAMPLE 1 Selection and Synthesis of Antigenic Peptides

Sequence Selection.

Three criteria were used to select sequences for putative antigenicpeptides: hydrophilicity, flexibility, and secondary structure.Prediction of hydropathy plots (Kyte, J. and Doolitle, R. F. J. Mol.Biol. 157:105-132, 1982) and secondary structure (Chou, P. Y. andFasman, G. D. Ann.Rev. Biochem. 47:251-276, 1978) of proteins wasaccomplished using computer analysis (PROSIS; Hitachi SoftwareEngineering Co., Ltd.). Segmental flexibility of proteins along a chainwas analyzed by a method described by Karplus and Schulz(Naturwissenschaften 72:212-213, 1985). In addition, the secondarystructure of proteins was predicted by the method developed by Ptitsinand Finkelstein (Biopolymers 22:15-25, 1983).

Sequences of the synthetic peptides selected to be synthesized are shownin Tables 1 and 2. Most of the peptides chosen for synthesis containhydrophilic and flexible sequences represented in the predictedsecondary structure as beta-turns or random coils (Tables 1 and 2).Synthetic peptides covering almost the entire ORF3 protein were preparedwith the exception of two very strong hydrophobic regions in theN-terminal half of the molecule. Using additional criteria (Eisenberg etal. J. Mol.Biol. 179:125-142, 1984; Klein et al. Biochem. Biophys.Acta815:468-476, 1985), these two regions may be predicted to betransmembrane alpha-helices. Hydropathy plots are important in selectingantigenic peptides, because hydrophobic regions usually have thepotential to be represented as α-helices and may function astransmembrane regions that may not be available for interaction with theimmune system. Strong hydrophobic potential of the N-terminal regionsuggests the protein could be associated with cellular membranespossibly playing a role in excretion of the HEV particles. Although HEvirions contain no known traces of lipids, existence of a stronghydrophobic region at the N-terminus of the structural protein(s)encoded by the large ORF2 additionally indicates a possible role ofmembranes in the morphogenesis of HEV. Taking this finding intoconsideration, a short sequence between these two hydrophobic regionswas selected for synthesis in addition to peptides spanning theC-terminal relatively hydrophilic region of the ORF3 protein. This shorthydrophilic sequence may be a loop located between these twotransmembrane domains. If this region is exposed, it could be a strongantigenic epitope of the ORF3 protein.

Due to the length of ORF2, synthetic peptides were selected usingadditional considerations. The whole N-terminal half of the proteincontains high concentrations of Arg and Lys. These amino acids have apotential to be bound to nucleic acids suggesting that in virionparticles this region may have an internal localization. Nonetheless, afew peptides were selected from the hydrophilic and flexible region ofthe N-terminal half of the protein for synthesis with the knowledge thatinternal proteins of viral particles such as HBV, HDV, or HCV have beenimportant diagnostic reagents.

Synthesis of Peptides.

Peptides were synthesized by FMOC-chemistry (Barany and Merrifield,1980) on an ABI Model 430A automated peptide synthesizer (AppliedBiosystems, Inc., Foster City, Calif.) or on an ACT Model MPS 350multiple peptide synthesizer (Advanced Chemtech, Louisville, Ky.)according to the manufacturer's protocols. Protocols have been describedpreviously and are available as FastMoc™ cycles (Applied Biosystems,Inc. 1990, FastMoc™ chemistry: HBTU activation in peptide synthesis onthe Model 430A Peptide Synthesizer User Bulletin 32; and HBTU activationin peptide synthesis on the Model 431A Peptide Synthesizer User Bulletin33). There are two scales, 0.10 and 0.25 mmol, with total cycle times of20 and 60 min, respectively. The Fmoc deprotections were done withpiperidine in N-methylpyrrolidone (NMP), and the resin was washed withNMP. In both scales, 1.0 mmol of Fmoc-amino acid was dissolved in NMP,and 1 mmol of 0.45 M HBTU(2-(H-benzotriazol-1-yl)-1,1,3,3,-tetramethyluronium hexafluorophosphate)/0.45 MHOBt(1-Hydroxybenzotriazole) in DMF(N,N-dimethylformamide). Thedissolved Fmoc-amino acid was transferred to the reaction vessel alongwith 1.7-2.0 mmol of DIEA(diisopropylethylamine) and allowed to reactwith the peptide resin for approximately 10 min with the 0.10 mmolcycles and 30 min with the 0.25 mmol cycles.

Peptides were cleaved by placing 10 mg of peptide resin in a test tubecontaining 200 μl of either 5% thioanisole/2.5% ethanedithiol/2.5% H₂O/90% TFA or 2.5% ethanedithiol/5% H₂ O/92.5% TFA and mixing for 1 h.Approximately 3.5 ml of H₂ O were then added, the mixture was extracted3-5 times with 2-3 ml of t-butyl methyl ether, and the aqueous layer wasfiltered. Any insoluble peptide was dissolved in the aqueous layer byaddition of acetic acid, ammonium hydroxide or isopropanol, asappropriate before analyses.

After characterization by amino acid analysis, high performance liquidchromatography and capillary electrophoresis, peptides were useddirectly in enzyme immunoassay.

Antigenic Activity of the Peptides.

Initial analysis of the peptides (Tables 1 and 2) utilizing enzymeimmunoassay (EIA) was performed on sera obtained from an outbreak inCentral Asia (Table 3). All the samples from this collection were testedby western blot analysis recently developed for HEV infection and by thepeptide EIA.

Sera.

Sera from outbreaks of enterically transmitted non-A, non-B hepatitis inCentral Asia (1985), Mexico (1986), and Kenya (1991) were used toidentify HEV-specific epitopes in proteins encoded by ORF2 and ORF3. Asa negative control for this analysis, a collection of sera from humanswith HAV, HCV, HBV, HDV markers of infection, or from normal blooddonors were used.

Sera from viral hepatitis patients ages 1 to 67 years were collectedfrom outbreaks of viral hepatitis between 1984 and 1992 ingeographically distinct regions of the world (Central Asia and Mexico).Whenever possible sera was obtained from infected patients beginning atthe onset of jaundice through the 27th day of jaundice. In addition,convalescent sera was obtained from 1-24 months after hospitaldischarge. All sera were originally diagnosed as NANBH by serologicexclusion of markers for HAV, HBV, and HCV using commercially availabletests (Preparat, Nizny Novgorod, Russina; Abbott Laboratories, NorthChicago, Ill.). Reagents: Affinity purified goat anti-human IgGconjugated to horseradish peroxidase (HRP) was obtained from TAGO(Burlingame, Calif.) and used at a dilution of 1:40,000. To confirm thespecificity of this detector molecule, monoclonal antibody specific forhuman IgG or for IgM conjugated to HRP was used at a dilution of 1:2000and

Western Blot Assay.

All sera were tested for anti-HEV activity by western blot that wascarried out according to Favorov et al. (J. Med. Virol. 36:246-250,1992).

Recombinant chimeric proteins, C2 and C2-1, containing the N-terminalregion of trypE (37 kDa) and the C-terminal half (46.8 kDa) of thepolypeptide encoded by ORF2 (Purdy et al., 1992) were used for thedevelopment of a Western blot (WB) diagnostic test to detect IgG and IgMclass antibodies to the HEV.

Recombinant fusion protein C2 (83.8 kDa) and the fusion protein C2-1(49.5 kDa) were expressed in Escherichia coli and lysates containingthese proteins were obtained as described previously (Purdy et al.,1992). C2 and C2-1 lysates were mixed and separated electrophoreticallyin 8% or 11% sodium dodecyl sulfate (SDS)-polyacrylamide gels (Purdy etal., 1992). Separated proteins were transblotted to BAS 83nitrocellulose (Scheicher & Schuell, Keene, NH) using a TE70 SemiPhor(Hoefer, San Francisco, Calif.) as described previously (Purdy et al.1992). Two millimeter strips were cut and incubated for 16-20 hr at 20°C. in 50 mM Tris-HCl, pH 7.5, containing 5 mM EDTA, 150 mM NaCl, 0.05%NP-40, 0.25% gelatin, and 1.0% bovine serum albumin (NET-BSA) prior tostorage at 4° C. Strips could be stored in NET-BSA for 1-2 weeks at 4°C. or dried under vacuum and stored for longer periods.

Unknown serum specimens were diluted (minimum dilution 1:20) in NETbuffer containing a 1% extract of an E. coli lysate without therecombinant plasmid and incubated for 2 hr at room temperature.Following incubation, the strips were washed in NET buffer three timesfor 10 min, and incubated with horseradish peroxidase (HRP) conjugatedaffinity purified detector antibodies. These included goat anti-humanIgM and goat anti-human IgG (TAGO, Burlingame, Calif.; Sigma ChemicalCo., St. Louis, Mo.). The specificity of human immunoglobulin isotypeswas confirmed with HRP conjugated murine monoclonal anti-human IgG andanti-human IgM (American Qualex, La Mirada, Calif.). The strips werethen incubated in phosphate-buffered saline (PBS), pII 7.2, containing100 mg of 3,3'-diaminobenzidine and 100 μl of H₂ O₂ for colordevelopment.

To eliminate non-specific reactions, C2 and C2-1 proteins wereindividually located and excised from the polyacrylamide gel. These wereeluted separately in 0.5 M Tris-HCl buffer, pH 6.8, electrophoreticallyrepurified in 8.5% SDS-polyacrylamide gels, transblotted, and testedwith the serum specimens that gave non-specific reactions. In addition,to confirm conjugate immunoreactivity for each analysis, purifiedimmunoglobulin heavy chains (gamma-specific and mu-specific, Chemicon,El Segundo, Calif.) were electrophoretically separated, transblotted,and immunostained separately.

This method for detecting HEV infection is not sensitive enough todetect a significant number of instances of acute or prior infection.Furthermore, the western blot method described is time consuming,because purification of the recombinant proteins is required before use.

Enzyme Immunoassay.

Microliter wells (Immulon I, Dynatech Laboratories, Inc.) were adsorbedwith 100 ul of synthetic peptides at a concentration of 5 ug per well.Human sera were diluted 1:50 in 0.1M PBS, pH 7.5, containing 0.1% Tween20 and 10% normal goat serum, and 100 ul were added to each well tocapture antibodies reactive to the adsorbed peptides. To identify seracontaining anti-peptide activity, affinity purified anti-humanantibodies conjugated to horseradish peroxidase (HRP) (heavy-chainspecific; TAGO, Inc., Burlingame, Calif., USA) were used.

The cutoff was statistically established to be three times the averagenegative control value.

Central Asia Epidemic

As shown in Table 3, four of 5 ORF3 specific peptides (2, 3, 5 and 6)and 4 of 12 ORF2 specific peptides (11, 12, 22 and 23) were reactivewith most of the sera. All but one sera positive by western blotanalysis were reactive in the EIA with one or more synthetic peptides.Peptides 5 and 6 seem to represent dominant antigenic epitopes, sincethese two peptides bind antibodies from almost all sera analyzed(97.5%). Only 1 of 40 western blot anti-HEV positive sera was notreactive with these peptides (Table 3).

Peptides 2, 3, 11, 12, 22 and 23 were reactive with approximately 30% ofsera. Peptides 10, 13, and 14 (Table 3) displayed reactivity with bothanti-HEV positive and negative sera. These peptides were considerednon-specific. Thus, 8 immunoreactive regions (peptides 2, 3, 5, 6, 11,12, 22 and 23) encoded by the large ORF2 and the small ORF3 wereidentified (Table 3).

Peptides 33 and 40 (Table 2) are also very immunoreactive. Peptide 33reacted with 81% of the anti-HEV positive sera (data not shown). Peptide40 is reactive with 45% of positive sera (data not shown). Both peptidesare very important for the diagnosis of HEV infection. These twopeptides can be included in the mixtures of the present invention (forexample, mixture 9) to improve the sensitivity of the peptide EIA forthe detection of an anti-HEV antibody.

Mexico Epidemic.

Synthesis of the majority of the present peptides, including 5 and 6,was based on the sequence of the Burma strain of HEV. Peptides 28 and 29were selected and synthesized based on the Mexico strain sequence(Yarbough et al., 1991) at precisely the same location as 5 and 6, butwith different sequences. Comparative analysis of peptides and 6, and 28and 29 demonstrated that there was no strict strain specificity forthese peptides (Table 4).

Peptides 28 and 29 are reactive with sera from the Mexico outbreak in1986 as expected; however, even combinations of peptides 5, 6, 22, 23,28 and 29 did not identify all sera positive by western blot analysis orby fluorescent antibody blocking assay (Krawczynski and Bradley, 1989)from the Mexico outbreak (Table 4, sample 395). In the IFA, knowninfected tissue was contacted with unknown serum, washed,detector-labeled human anti-HEV IgG added, and observed for the presenceof label. The same specimen (395) did not react with any peptide,however, it remains strongly reactive by western blot analysis and byIFA. Additionally, specimen 67 (Table 4) was anti-HEV negative bywestern blot analysis and IFA, but was reactive with peptides 5, 6 and22.

Peptides 5 and 6 always reacted with the same set of sera; however,peptides 28 and 29 having the same location but based on the Mexican HEVstrain sequence did not always react simultaneously with the samespecimen. An epitope represented in peptide 28 appeared to be moreimmunoreactive than an epitope represented in peptide 29 (Table 4).Thus, there appears to be a difference in the primary structure ofpeptides 5 and 28, and 6 and 29 that may lead to changes in antigenicactivity of these epitopes.

Kenya Epidemic.

When analyzing sera obtained from a recent outbreak in Kenya peptides 5,6, 22, 23, 28, and 29 were tested against the sera. Among 93 sera chosenfor peptide analysis, 24 sera were obtained from patients with reportedjaundice within half a year (Table 5). The remaining 69 specimens werecollected from people without a history of jaundice, but who lived inthe same location where the outbreak occurred (Table 6). Initially, allsera were characterized by western blot analysis for anti-HEV activityand for the presence of IgM antibody to the hepatitis A virus (HAV) byEIA. All sera were negative for IgM anti-HAV activity (not shown).

Among sera from reported cases of jaundice, 23 were found anti-HEVpositive by peptide EIA. Twenty-seven sera were found anti-HEV positiveby western blot analysis among specimens obtained from individuals whohad not reported jaundice within the last 6 months (Table 6).

Peptide 29 was found non-reactive with these sera from Kenya and peptide28 was reactive with only a few samples. However, peptides 5 and 6reacted with about 71% of sera from the reported cases of jaundice and48.2% of sera found anti-HEV positive by western blot analysis. Onespecimen (138) from an individual with no history of jaundice was foundspecifically reactive with peptides 5 and 6 but not reactive by westernblot analysis.

Peptides 22 and 23 were more reactive than 5 and 6. More than 95% ofsera from patients with reported cases of jaundice was identified withboth of these peptides. About 82% of sera from people with no history ofjaundice positive by western blot analysis were also reactive with thesepeptides. Eight sera negative by western blot analysis were found tocontain antibody activity specifically reactive with peptides 22 and 23.Thus, a combination of peptides 22 and 23 identified more anti-HEVpositive sera than peptides 5 and 6. Individual peptides 22 and 23 usedseparately demonstrated stronger immunoreactivity than peptides 5 and 6.For example, peptide 22 reacted with 79.2% and 66.7% of sera, andpeptide 23 with 79.2% and 63% of sera obtained from people who hadreported jaundice or positive by western blot analysis for anti-HEVactivity, respectively (compared with 70.8% and 48.2% for 5 and 6mentioned above). Collectively, peptides 5, 6, 22, and 23 could identifyall reported cases of HEV infection from the outbreak in Kenya in thesynthetic peptide EIA. Western blot analysis was less sensitive comparedwith peptide EIA. Only one specimen (144) obtained from patients withjaundice was not identified as positive by western blot analysis (Table5).

Many sera containing anti-HEV activity without jaundice may be due toeither past HEV infections or due to asymptomatic HEV infections. Amongreported cases with jaundice there were more examples ofimmunoreactivity against 4 different peptides than among cases without ahistory of disease. Peptides 5, 6 and 22, and 23 identified the samepercent of patients among reported cases of the disease. However,peptides 5 and 6 were 2-fold less reactive with sera from the group notreported as jaundice cases (see Table 6). The humoral immune response tothe antigenic epitopes of peptides 5 and 6 may decay faster than theepitopes of peptides 22 and 23. Thus peptides 5 and 6 can be used forthe differential diagnosis of various stages of the HEV infection.Conversely, peptide 13 can detect antibodies in serum up to 3 yearsafter acute infection.

Strain specificity is shown insofar as sera obtained from Mexico have adifferent pattern of interaction with the synthetic peptides comparedwith sera obtained from Central Asia or from Kenya (Table 3-6). There isonly a 78% homology between the ORF3 protein region comprising 5 and 6,or 28 and 29 (Yarbough et al., 1991). With the Mexico sera, peptides 5and 6 identified the same percent of positives as 28 and 29 (Table 2).However, with sera from Central Asia (data not shown) and from the Kenyaoutbreak, peptides 28 and 29 were less reactive as evidenced by a lowpercentage of sera positive for anti-HEV using these peptides. Thus, ourdata shows that the Burma strain of HEV is related to a greater extentto the Kenya or Central Asia viruses than to the Mexico strain of HEV.

There are at least 4 antigenic regions in the protein encoded by ORF3and at least 7 antigenic regions in the protein encoded by ORF2. Among12 peptides selected for synthesis from the structural protein(s) only 4(33%) were specifically reactive with sera from HEV infectedindividuals.

Identification of Minimal Antigenic Sequence.

A set of peptides of 6 amino acids long (or 7, or 8 etc.) must besynthesized. These peptides are overlapped by 5 aa long regions (or 6for 7 aa long peptides, etc.) and collectively contain the sequencewhere the antigenic epitopes have been already found (for example,peptide 5, or 6 or 22, or 23, or 13, etc.). Antigenic activity of eachshort peptide from the set is analyzed by the procedures describedherein. Immunoreactive regions of 6 aa long (or 7 aa, or 8 aa, etc.) areused to design other peptides of 5 aa long which are overlapped by 4 aaregions. If the antigenic activity remains in the peptides, a set ofshorter peptides of 4 aa long is synthesized. All peptides arecomparatively analyzed to find the best peptide sequences strongly andspecifically immunoreactive with anti-HEV antibody. These peptides couldalso be used in a mixture to detect anti-HEV antibody.

                  TABLE 1                                                         ______________________________________                                        PRIMARY AND PREDICTED SECONDARY STRUCTURE FOR                                 SELECTED REGIONS OF THE PROTEIN ENCODED BY                                    ORF2 OF HEV GENOME                                                            Pep-          Primary and secondary                                           tide Position structure                                                       ______________________________________                                         7    54-65   PYIHPTNPFAPD      (SEQ ID NO:14)                                              sssstttttccc                                                     9    84-101  GSAWRDQAQRPAVASRRR                                                                              (SEQ ID NO:15)                                              ccccccccccttcccccc                                              19   312-329  TPGNTNTRVSRYSSTARH                                                                              (SEQ ID NO:20)                                              ctttcccccccccccccc                                              11   319-340  RVSRYSSTARHRLRRGADGTAE                                                                          (SEQ ID NO:5)                                               ccccccccccccccccccccc                                           33   415-433  TSVENAQQDKGIAIPHDIDL                                                                            (SEQ ID NO:12)                                              sssttttsssssssstttss                                            12   422-437  DKGIAIPHDIDLGESR  (SEQ ID NO:6)                                               cttssstttccttttc                                                13   442-460  DYDNQHEQDRPTPSPAPSR                                                                             (SEQ ID NO:7)                                               ccccccccccccccccttc                                             14   479-492  EYDQSTYGSSTGPV    (SEQ ID NO:16)                                              cccccctttcttts                                                  15   521-534  LDGRPLSTIQQYSK    (SEQ ID NO:17)                                              sccctttccctttt                                                  17   612-634  DTLDYPARAHTFDDFCPECRPLG                                                                         (SEQ ID NO:18)                                              ccccctttcccccccctttcttt                                         40   562-580  NTTASDQLLVENAAGHRVA                                                                             (SEQ ID NO:13)                                              sstttcsssssttttccss                                             22   631-648  RPLGLQGCAFQSTVAELQ                                                                              (SEQ ID NO:8)                                               ctttccccccccchhhhh                                              23   641-660  QSTVAELQRLKMKVGKTREL                                                                            (SEQ ID NO:9)                                               ccchhhhhhhcccccccccc                                            ______________________________________                                         Elements of secondary structure are indicated as follow:                      halpha-helix; sbeta-sheet; tbeta-turn; crandom coil                      

                  TABLE 2                                                         ______________________________________                                        PRIMARY AND PREDICTED SECONDARY STRUCTURE FOR                                 THE SELECTED REGIONS OF THE PROTEIN ENCODED BY                                ORF3 OF HEV GENOME                                                            Pep-          Primary and secondary                                           tide Position structure                                                       ______________________________________                                         2    31-40   CPRHRPVSRL        (SEQ ID NO:3)                                               stttctttcs                                                       3    63-76   SPSQSPIFIQPTPSG   (SEQ ID NO:4)                                               stttsssssccccc                                                   4    73-87   PTPSPPMSPLRPGLD   (SEQ ID NO:19)                                              cccctttcttcttss                                                  5    91-110  ANPPDHSAPLGVTRPSAPPLA                                                                           (SEQ ID NO:1)                                               ccttcccctttcccttcccc                                             6   105-123  PSAPPLPHVVDLPQLGPRR                                                                             (SEQ ID NO:2)                                               ttcccccccccctttcccc                                             28    91-110  ANQPGHLAPLGEIRPSAPPLA                                                                           (SEQ ID NO:10)                                              ccttcccctttcccttcccc                                            29   105-123  PSAPPLPPVADLPQPGLRR                                                                             (SEQ ID NO:11)                                              ttccccccccccttccccc                                             ______________________________________                                         Elements of secondary structure are indicated as follow:                      halpha-helix; sbeta-sheet; tbeta-turn; crandom coil\                          Peptides 28 and 29 represent the protein encoded by ORF3 of Mexico strain     HEV (Yarbough et al., 1991).                                             

                                      TABLE 3                                     __________________________________________________________________________    IDENTIFICATION OF SYNTHETIC PEPTIDES SPECIFICALLY REACTIVE WITH               SERA FROM PATIENTS INFECTED WITH HEPATITIS E VIRUS                            (Central Asia, 1985)                                                          SERA POSITIVE BY WESTERN BLOT                                                                            NEGATIVE SERA                                      PEPTIDE                                                                            TOTAL                                                                             +++                                                                              ++ +  W+ -  (+) %                                                                            TOTAL                                                                             +  -                                           __________________________________________________________________________     2   33  2  3  5  0  23 30.3                                                                             26  1  25                                           3   32  2  1  5  1  23 28.1                                                                             27  0  27                                           4   12  0  0  0  0  12 0  9   0  9                                            5   40  25 9  1  4   1 97.5                                                                             27  0  27                                           6   40  24 9  0  6   1 97.5                                                                             27  0  27                                           7    5  0  0  0  0   5 0  3   0  3                                            9   13  0  0  0  0  13 0  9   0  9                                           10   13  0  0  0  1  12 NS 9   1  8                                           11   33  2  3  3  2  23 30.3                                                                             26  1  25                                          12   39  3  3  4  2  27 30.8                                                                             26  0  26                                          13   13  0  1  0  0  12 NS 8   3  5                                           14    8  0  0  0  1   7 NS 12  1  11                                          15   12  0  0  0  0  12 0  9   0  9                                           17   25  0  0  0  0  25 0  18  0  18                                          19   13  0  0  0  0  13 0  9   0  9                                           22    7  2  0  0  0   5 28.6                                                                             5   0  5                                           23    7  2  1  0  0   4 42.9                                                                             5   0  5                                           __________________________________________________________________________     POSITIVES:                                                                    +++ with P/N >10                                                              ++ with P/N = 5-10                                                            + with P/N = 3-4.9                                                            W+ with P/N = 2.1-2.9                                                         NS  nonspecific                                                          

                  TABLE 4                                                         ______________________________________                                        SEROLOGIC ASSAY FOR THE DETECTION OF HEV INFECTION                            (Mexico, 1986)                                                                PEPTIDE EIA                                                                   SAMPLE 5      6      22   23   28   29   IFA (*)                                                                             W/B                            ______________________________________                                         6     +++    +++    ND   ND   ND   ND   +     +                               7     +++    +++    ND   ND   ND   ND   +     +                              11     -      -      -    -    -    -    -     -                              15     +      +      -    +++  +    -    +     +                              19     +++    +++    +++  +++  +++  -    +     +                              66     +      +      ++   ++   +    +    +     +                              67     ++     ++     +    -    -    -    -     -                              68     -      -      -    -    -    -    -     -                              73     +++    +++    +    +++  +++  +    +     +                              75     +      +      +    ++   +    +    +     +                              390    +++    +++    -    -    -    -    +     +                              395    -      -      -    -    -    -    +     +                              397    +      +      +    +++  +++  +++  +     +                              399    -      -      -    -    +    +    +     +                              ______________________________________                                         POSITIVES:                                                                    +++ with P/N >10                                                              ++ with P/N = 5-10                                                            + with P/N = 3-4.9                                                            (*) Fluorescent antibody blocking assay                                  

                  TABLE 5                                                         ______________________________________                                        REPORTED CASES OF HEV INFECTION                                               (Kenya, 1991)                                                                 WESTERN BLOT    PEPTIDE EIA                                                   SAMPLE IgG      IgM     5    6    22  23   28   29                            ______________________________________                                        40     +        +       +    +    +   +    -    -                             55     +        +       +    +    +   +    -    -                             68     +        +       +    +    +   +    -    -                             69     +        -       +    +    +   +    -    -                             70     +        +       +    +    +   +    -    -                             71     +        -       +    +    -   +    +    -                             72     +        +       +    +    +   +    -    -                             74     +        +       +    +    +   +    +    -                             75     +        +       +    +    +   +    -    -                             83     +        +       +    +    +   +    -    -                             94     +        -       +    +    -   -    -    -                             95     +        -       +    +    -   +    -    -                             96     +        -       +    +    +   -    +    -                             97     +        +       +    +    +   -    -    -                             99     +        +       +    +    -   +    -    -                             100    +        +       -    -    +   +    -    -                             105    +        -       -    -    +   +    -    -                             116    +        +       +    +    +   +    -    ND                            122    +        +       -    -    +   +    -    ND                            128    +        +       -    -    +   +    -    ND                            144    -        -       -    -    +   -    -    ND                            110    +        +       ND   ND   +   -    +    ND                            161    +        +       +    +    -   +    -    ND                            TOTAL: 23 cases                                                               ______________________________________                                    

                  TABLE 6                                                         ______________________________________                                        SAMPLES POSITIVE FOR SEROLOGIC MARKERS OF HEV                                 INFECTION WITHOUT HISTORY OF JAUNDICE                                         (Kenya, 1991)                                                                 Western blot   PEPTIDE EIA                                                    Sample IgG     IgM     5    6    22   23   28   29                            ______________________________________                                         29    +       +       -    -    +    +    -    -                              30    +       -       +    +    +    +    +    -                              32    +       -       -    -    -    -    -    -                              35    +       -       -    -    +    +    -    -                              36    +       -       +    +    -    -    -    -                              37    +       +       -    -    +    +    -    -                              39    +       -       +    +    -    +    -    -                              47    +       -       -    -    +    +    -    -                              48    +       -       +    +    +    +    +    -                              50    +       -       +    +    -    -    -    -                              79    +       -       -    -    -    +    -    -                              80    +       +       -    -    -    +    -    -                              81    -       -       -    -    -    +    -    -                              82    +       -       +    +    -    -    -    -                              86    -       +       -    -    -    -    -    -                              87    -       -       -    -    +    +    -    -                              88    -       -       -    -    +    +    -    -                              93    +       +       -    -    +    +    -    -                             102    -       -       -    -    +    -    -    -                             103    +       +       -    -    +    -    -    -                             106    +       +       +    +    +    +    +    -                             107    +       +       +    +    +    -    -    ND                            113    +       +       -    -    +    +    -    ND                            115    +       -       -    -    +    +    -    ND                            117    +       -       -    -    -    -    -    ND                            118    +       -       +    +    +    -    -    ND                            119    -       -       -    -    +    +    -    ND                            123    +       -       -    -    +    +    -    ND                            124    +       +       -    -    +    +    -    ND                            133    -       +       -    -    +    -    -    ND                            138    -       -       +    +    -    +    -    ND                            140    +       +       -    -    -    +    -    ND                            142    +       +       +    +    +    +    -    ND                            146    +       +       +    +    +    +    -    ND                            149    -       -       -    -    +    -    -    ND                            152    +       -       +    +    +    -    -    ND                            159    +       -       +    +    +    -    -    ND                            total: 37 samples                                                             ______________________________________                                    

                  TABLE 7                                                         ______________________________________                                        Anti-HEV Activity by Peptide EIA                                              Percent positivity by region and source of specimen                                                           Anti-HEV                                      Region     Source of specimen                                                                         Total   positive No. (%)                              ______________________________________                                        Tadgikistan                                                                              outbreak     64      63                                                                            (98.4%)                                       Tadgikistan                                                                              healthy population                                                                         639     41                                                                             (6.4%)                                       Kergistan  outbreak     82      70                                                                            (85.4%)                                       Mexico     outbreak     38      32                                                                            (84.2%)                                       U.S.A. (sentinel                                                                         sporadic     39      0                                             counties)                                                                     Montana, USA                                                                             HAV/outbreak 283     1                                                                             (0.35%)                                       Russia     HBV          38      0                                             Russia     HDV          25      0                                             Russia     HCV          25      0                                             ______________________________________                                    

EXAMPLE 2 Enzyme Immunoassay for the Detection of Anti-HEV ActivityBased on Synthetic Peptides

Seventeen synthetic peptides based on the Burma strain of HEV andencoded in ORF2 and ORF3 were first used individually for theidentification of immunoreactive epitopes as described above (Table 3).Eight of these peptides contained important immunodiagnostic epitopeswhen tested against sera obtained from HEV infected individuals.Peptides 5 and 6 corresponding to the C-terminus of ORF3 identified morethan 90% of acute-phase sera (Table 3) and approximately 30% ofconvalescent-phase sera (data not shown). Other synthetic peptides whentested individually yielded lower rates of reactivity compared topeptides 5 and 6, or demonstrated non-specific reactions. Consequently,various mixtures of synthetic peptides were examined on the solid-phase.

To ascertain the diagnostic significance of various mixtures ofsynthetic peptides, each mixture was tested against a panel of sera.This panel was composed of 45 acute-phase specimens (1-10 days afteronset of jaundice) obtained from an HEV outbreak in an endemic region ofCentral Asia, and 14 follow-up convalescent-phase specimens (4-6 mosafter onset of jaundice) from these patients. In addition, 36 seraobtained from a normal donor population from a non-endemic region wereused as negative controls. Each specimen was diluted in normal goatserum buffer (NGS-Buff) composed of 0.01 M PBS, pH 7.2-7.4, containing10% normal goat serum (NGS), 1% bovine serum albumin (BSA), and 0.05%Tween 20. Anti-HEV activity in each member of the panel was ascertainedby WB analysis (Favorov et al. J. Med. Virol., 1992). All acute-phaseand convalescent-phase sera tested positive by WB, while all the donorsera were negative by WB.

The panel described above was not only used to assess differentcombinations of synthetic peptides adsorbed to the solid-phase, but alsoused to assess the sensitivity and specificity of the peptide-EIA byascertainment of positivity among the acute- and convalescent-phasesera, and among the normal donor population sera, respectively.

Three different approaches were used to configure various mixtures ofsynthetic peptides adsorbed to the solid-phase.

Unconjugated Peptide Mixtures.

The first approach (mixtures 1-5) was composed of only unconjugatedsynthetic peptides in various concentrations. The results of eachmixture were empirically obtained and mixture 5 adsorbed to thesolid-phase yielded the best results. This mixture was composed of thefollowing synthetic peptides: #6 (20 ug/ml), #5 (10 ug/ml), #11 (5ug/ml), #12 (1 ug/ml), #22 (1 ug/ml), #23 (5 ug/ml), #28 (2 ug/ml), and#29 (2 ug/ml). Peptides #28 and #29 are based on the Mexico strain ofHEV, as described above, and correspond to the exact nucleotide positionof peptides #5 and #6. The frequency of anti-HEV using mixture 5 was41/45 (91.1%) among acute-phase sera, 12/14 (85.7%) amongconvalescent-phase sera, 3/36 (8.3%) among normal donor sera. The 8.3%positivity value was considered the result of false-positive reactions.

Individual Peptides Conjugated to BSA.

The second approach involved conjugating BSA to individual syntheticpeptides. Peptides were conjugated to BSA using1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC)(Pierce Chemical Co., Rockford, Ill.) according to the method ofBauminger and Wilchek (15). Briefly, 10 mg of individual syntheticpeptide or a mixture of peptides was dissolved in distilled water at afinal concentration of 1 mg/ml. EDC crystals were added to the peptidesolution at a final concentration of 10 mg/ml and the pH was adjusted to5.0. The reaction mixture was incubated at room temperature for 5 minand BSA was added to yield a final molar ratio of approximately 1 moleof peptide to each 10 amino acids of BSA. The reaction mixture wasfurther incubated at room temperature for 4 hours. The conjugationreaction was stopped by adding sodium acetate, pH 4.2, to a finalconcentration of 100 mM and the reaction mixture was incubated at roomtemperature for an additional 1 hour. The peptide-protein conjugate wasseparated from the remaining peptide and other reaction products bydialysis against 0.01 M phosphate buffered saline, pH 7.2, overnight at4° C. The peptide-protein conjugate was stored in the same buffer at-20° C. This protocol will work for other peptides regardless ofsequence.

Conjugates were diluted in normal goat serum buffer (NGS-Buff) composedof 0.01 M PBS, pH 7.2-7.4, containing 10% normal goat serum (NGS), 1%bovine serum albumin (BSA), and 0.05% Tween 20. This buffer was alsoused for the development of a neutralization test (Neut-Buff).

After conjugation various concentrations of conjugated peptides(mixtures 1-7) were mixed and adsorbed to the solid-phase. The optimalresults were obtained with mixture 7 and was determined empirically.This mixture was composed of the following conjugated peptides: #23 (20ug/ml), #6 (14 ug/ml), #5(8 ug/ml), #22 (4 ug/ml), and #13 (4 ug/ml).The frequency of anti-HEV using mixture 7 was 44/45 (97.8%) amongacute-phase sera, 12/14 (85.7%) among convalescent-phase sera, and 1/36(2.8%) among normal donor sera. This specimen was considered afalse-positive reaction. Although peptide #13 demonstrated significantnon-specific reactions when tested unconjugated (Table 1), afterconjugation to BSA this non-specific reaction disappeared based oncomparison of mixtures 5 and 7.

Peptide Mixtures Conjugated to BSA.

The third approach involved conjugating a mixture of the sameconcentrations of synthetic peptides as indicated above in mixture 7 toBSA (mixture 9). In this way, theoretically, each epitope wasrepresented on each molecule of BSA and resulted in the synthesis of anartificial antigen complex that may approximate the immunoreactivity ofthe natural antigen. In addition, such a complex may allow for a moreuniform distribution of antigenic epitopes on the solid-phase. Usingmixture 9, 45/45 acute-phase sera were positive for anti-HEV activity,13/14 convalescent-phase sera were positive, while all normal donor seraremained negative.

Peptide-EIA.

Immulon II EIA microtiter wells (Dynatech Laboratories, Inc. Chantilly,Va.) were adsorbed with 105 ul of individual peptides or conjugatedpeptides overnight at room temperature. After adsorption, each well waswashed 5 times with deionized H2O containing 0.5% Tween 20. Eachspecimen was then diluted appropriately in NGS-Buff and 100 ul of thediluted specimen was added to each well. Following an incubation periodof 1 hr at 37 C., each well was washed 7 times, and 100 ul of dilutedconjugate was added to each well. The wells were then incubated againfor 1 hr at 37 C., washed 7 times, and 100 ul of substrate solution(o-phenylenediamine and H2O2 obtained from Abbott Laboratories) wasadded. The wells were incubated for 15-30 min at room temperature in thedark, after which the enzyme reaction was stopped with 50 ul of 1 NH2SO4. The wells were read in an ELISA reader set at an optical densityof 493 nm. The cutoff value for a positive result was statisticallydetermined based on a frequency distribution of 480 WB positive andnegative sera.

To assess the individual antigenic immunologic activity of three out offive epitopes in mixture 9 adsorbed to the solid-phase, serial 2-folddilutions of individual guinea pig anti-peptide #23, #6 and #5 wereadded to the solid-phase and incubated. After a wash cycle, goatanti-guinea pig IgG conjugated to HRP was used to detect antigenicepitope activity on the solid-phase. As a control, guinea piganti-peptide #28 was similarly diluted and tested with mixture 9, whichis devoid of peptide #28 activity. The endpoint of each anti-peptideserum exceeded a dilution of 1:32,000 indicating that these epitopeswere immunologically active and accessible in mixture 9 after adsorptionto the solid-phase, while the negative control remained unreactive ateach dilution.

Frequency Distribution.

To determine the utility of the peptide-EIA to discriminate betweenpositive and negative sera, 483 sera were selected and tested at a 1:100dilution against mixture 9 and a frequency distribution was constructed(FIG. 2). The frequency distribution revealed that three zones could bedefined; namely, a positive zone, a negative zone, and an indeterminatezone. The cutoff for the positive zone was equal to the mean of negativecontrols plus 6.3 standard deviations (SD) of the mean. Optical densityvalues below 0.07 (mean plus 3.9 SD or mean times 3.2) were consideredin the negative zone, while optical density values greater than 0.07 andless than 0.10 were considered in the indeterminate zone. Threespecimens yielded indeterminate values. Upon retesting at a 1:10dilution, these sera tested positive for anti-HEV activity.

Neutralization Assay.

A neutralization test was developed to confirm anti-HEV activity insera. Briefly, follow the above protocol for peptide EIA, but incubatethe specimen with mixture 5 (individual unconjugated peptides) beforeadding to mixture 9; remove the specimen incubated with mixture 5 andadd it to absorbed mixture 9. This test is particularly important forthose specimens that yielded optical density values close to the cutoffvalue and for those specimens that yielded optical density values withinthe indeterminate zone. The use of a neutralization test significantlylowered the false-positivity rate, especially in sera which have beenimproperly stored or which have undergone several freeze-thaw cycles.The neutralizing agent must be different from the peptide or peptidemixture adsorbed to the solid-phase; otherwise, false-positive reactionswould be incorrectly neutralized leading to confirming a truly negativespecimen as being positive. Thus, individual synthetic unconjugatedpeptides were used as the neutralizing agent by incubating the dilutedspecimen at 37° C. for 1 hr in a fluid-phase before addition to the wellcontaining adsorbed mixture 9. After testing various concentrations, theoptimal concentration of each synthetic unconjugated peptide was asfollows: #23 (40 ug/ml), #6 (30 ug/ml), #5 (15 ug/ml), #22 (10 ug/ml),#13 (10 ug/ml).

FIG. 3 presents the results of a neutralization test using sera whichinitially tested positive and yielded optical density values from 0.1 togreater than 2.5. Sera were confirmed as positive when the opticaldensity values were decreased by 50% following incubation with theneutralizing mixture. With the exception of sera that gave OD valuesgreater than 2.5, all sera were confirmed as positive with meanneutralization activities ranging from between 63% to 78%. Sera thatyielded OD values greater than 2.5 were retested at a higher dilution atof 1:500. All sera were successfully neutralized after dilution.

The final design of the peptide-EIA involves testing each specimen foranti-HEV activity simultaneously with and without neutralization at aninitial dilution of 1:50. The first row of the microtiter wells containssera diluted in NGS-buff and the second row contains diluted andneutralized sera so that the same sera are located in two wells in avertical position. Initially reactive but non-neutralized sera whichyielded OD values greater 2.0 were retested at a 1:100 dilution. Inaddition, sera that yielded OD values greater than 2-times the mean ofnegative controls and that were not neutralized were retested at a 1:10dilution. These sera were considered to be positive for anti-HEVactivity if the OD values were reduced by 50% following neutralization.

Endpoint Determinations.

Following the final design of the peptide-EIA, endpoint determinationswere made on thirty-three sera with OD values greater than 2.0. Most ofthese sera demonstrated an endpointtiter of 1:1000 to 1:10,000. Threesera within this group demonstrated endpoint titers exceeding 1:100,000(data not shown).

Anti-HEV Activity by Region and Source.

The peptide-EIA using mixture 9 was used to determine anti-HEV activityin sera collected from various regions of the world and included serafrom HEV outbreaks in two HEV endemic areas (Tadgikistan and Kergizstan,central Asia republics, former USSR; and Mexico), sera from a healthypopulation obtained from an HEV endemic region (Tadgiskistan), and serafrom an HEV non-endemic area (USA) (Table 7). The Montana collectionrepresented a case controlled investigation of an HAV outbreak. Inaddition, acute-phase sera obtained from Russia positive for markers ofHBV, hepatitis delta virus (HDV), and HCV were used as controls. None ofthe controls demonstrated anti-HEV activity indicating that thepeptide-EIA did not falsely identify these sera as past HEV infections.Only one serum in the Montana collection was repeatedly positive(confirmed by neutralization) for anti-HEV activity and remainedpositive when a follow-up specimen was obtained two years later. Thesentinel county specimens represent acute-phase sera collected from anactive surveillance system implemented in 4 geographically distinctcounties in the USA. The 39 sera were diagnosed as NABCH without anyevidence of chronic sequelae on follow-up. All of these sera werenegative for anti-HEV activity indicating, with the exception of thesingle positive serum in Montana, HEV infection in the USA is a rareevent, excluding infections acquired upon travel to an endemic region ofthe world.

Among 639 sera collected from healthy individuals in an HEV endemicregion, 41 (6.4%) demonstrated anti-HEV activity indicating priorexposure to HEV. This value represents HEV background of infection in ahealthy population in an endemic region of the world.

Finally, in two outbreaks of HEV in the central Asian republic of theformer USSR, Tadgiskistan and Kergistan, 98.4% and 85% of the sera werepositive for anti-HEV activity, respectively. Collectively, these datashow that the peptide-EIA for the detection of anti-HEV is highlyspecific and sensitive.

Throughout this application various publications are referenced. Thedisclosures of these publications in their entireties are herebyincorporated by reference into this application in order to more fullydescribe the state of the art to which this invention pertains. Anyincomplete citations referenced above can be found in the followingreference list.

REFERENCES

Balayan M S, Andjaparidze, Savinskaya S S, et al. Evidence for a virusin non-A, non-B hepatitis via the fecal-oral route. Intervirology1983;20:23-31.

Barany, G., and Merrifield. (1980). Solid-phase peptide synthesis. In"The Peptides" (E. Gross and J. Meienhofer, Eds.) 1, 1-284. AcademicPress, NY.

Bradley D W, Krawczynski K, Cook E H, et al. Enterically transmittednon-A, non-B hepatitis: serial passage of disease in cynomolgus macaquesand tamarins and recovery of disease-associated 27- to 34-nm viruslikeparticles. PNAS USA 1987;84:6277-6281.

Bradley, D. W. (1990a). Hepatitis non-A, non-B viruses become identifiedas hepatitis C and E viruses. Prog. Med.Virol. 37, 101-135.

Bradley, D. W. (1990b). Enterically-transmitted non-A, non-B hepatitis.British Medical Bulletin 46, 442-461 CDC (1987a). Entericallytransmitted non-A, non-B hepatitis--East Africa. MMWR 36, 241-244. CDC(1987b). Enterically Transmitted non-A, non-B hepatitis - Mexico. MMWR36, 597-602.

Bauminger S, Wilchek M. The use of carbodiimides in the preparation ofimmunizing conjugates. Methods Enzymol 1980;70:151-159.

Chou, P. Y., and Fasman, G. D. (1978). Empirical predictions of proteinconformation. Ann. Rev. Biochem. 47, 251-276.

Eisenberg, D., Schwarz, E., Kamaromy, M., and Wall, R. (1984). Analysisof membrane and surface protein sequences with the hydrophobic momentplot. J. Mol. Biol. 179, 125-142.

Favorov, M. O., Fields, H. A., Purdy, M. M. et al. (1992). Serologicidentification of hepatitis E virus infections in epidemic and endemicsettings. J. Med. Virol. 36, 246-250.

Goldsmith, R., Yarbough, P. O., Reyes, G. R., et al. (1992).Enzyme-linked immunosorbent assay for diagnosis of acute sporadichepatitis E in Egyptian children. Lancet 339, 328-331.

Harada, S., Watanabe, Y., Takeuchi, K. et al. (1991). Expression ofprocessed core protein of hepatitis C virus in mammalian cells. J.Virol. 65, 3015-3021.

Ichikawa, M., Araki, M., Rikihisa, T. et al. (1991). Cloning andexpression of cDNAs from enterically-transmitted non-A, non-B hepatitisvirus. Microbiol.Immunol. 35, 535-543.

Karplus, P. A., and Schulz, G. E. (1985). Prediction of chainflexibility in proteins: a tool for selection of peptide antigens.Naturwissenschaften 72, 212-213.

Khuroo M S. Study of an epidemic on non-A,non-B hepatitis: possibilityof another human hepatitis virus distinct from post-transfusion non-A,non-B type. Am J Med 1980;68:818-824.

Klein, P., Kanehisa, M., and DeLisa, C. (1985). The detection andclassification of membrane-spanning proteins. Biochem. Biophys. Acta815, 468-476.

Krawczynski, K., and Bradley, D. W. (1989). Enterically transmittednon-A, non-B hepatitis: Identification of virus-associated antigen inexperimentally infected cynomolgus macaques. J. Infect. Dis. 159,1042-1049.

Kyte, J. and Doolittle, R. F. (1982). A simple method for displaying thehydropathic character of a protein. J. Mol. Biol. 157, 105-132.

Ptitsyn, O. B., and Finkrlstein, A. V. (1983). Theory of proteinsecondary structure and algorithm of its prediction. Biopolymers 22,15-25.

Purcell R H, Ticehurst J R. Enterically transmitted non-A, non-Bhepatitis: epidemiology and clinical characteristics. In: Zuckerman A J,ed. Viral hepatitis and liver disease. New York: Alan R.Liss,1988:131-137.

Purdy, M., McCaustland, A., Krawczynski, K., et al. (1992). Expressionof a hepatitis E virus (HEV)-trpE fusion protein containing epitopesrecognized by antibodies in sera from human cases and experimentallyinfected primates. J. Arch. Virol. in press.

Reyes G R, Purdy M A, Kim J P et al. Isolation of a cDNA from the virusresponsible for enterically transmitted non-A, non-B hepatitis. Science1990;247:1335-1339.

Reyes, G. R., Huang, C. C., Yarborough, P. O., et al. (1991a). HepatitisE Virus (HEV): epitope mapping an detecting of strain variation. In"viral hepatitis C, D, E" (T. Shikata, R. H. Purcell, T. Uchida, Ess.),pp.237-245. Elsevier Science Publishers, NY.

Reyes, G. R., Yarbough, P. O., Tam, A. W., et al. (1991b). Hepatitis Evirus (HEV): The novel agent responsible for enterically transmittednon-A, non-B hepatitis. Gastroenterologia Japonica 26 (suppl.3),142-147.

Tam, A. W., Smith, M. M., Guerra, M. E., et al. (1991). Hepatitis Evirus (HEV): Molecular cloning and sequencing of the full-length viralgenome. Virology 185, 120-131.

Tassopoulos, N. C., Koutelou, M. G., Macagno, S. et al. (1990).Diagnostic significance of IgM antibody to hepatitis delta virusfulminant hepatitis B. J. Med. Virol. 30, 174-177.

Vishwanathan, R. (1957). Infectious hepatitis in Delhi (1955-56). IndianJ. Med. Res. (Suppl.) 45, 1-30.

Wang, Q. H., Lu, Z. M., Wang, Y. Q., and Chen, M. J. (1985). Diagnosticsignificance of IgM anti-HBc detection by ELISA in HBV infection. Chin.Med. J. 98, 703-707.

Wong D C, Purcell R H, Screenivasan M A, S R Prasad, K M Pavri. Epidemicand endemic hepatitis in India: evidence for a non-A, non-B hepatitisvirus aetiology. Lancet 1980; ii:876-879.

Wright, R. (1990). Viral hepatitis comparative epidemiology. BritishMedical Bulletin 46, 549-558.

Yarbough, P. O., Tam, A. W., Fry, K. E., et al. (1991). Hepatitis Evirus: Identification of type-common epitopes. J. Virol. 65, 5790-5797.

Zuckerman A J. Hepatitis E virus: the main cause of entericallytransmitted non-A, non-B hepatitis. Br Med J 1990;300:1475-1476.

    __________________________________________________________________________    #             SEQUENCE LISTING                                                - (1) GENERAL INFORMATION:                                                    -    (iii) NUMBER OF SEQUENCES: 20                                            - (2) INFORMATION FOR SEQ ID NO:1:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 21 amino                                                          (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                 - Ala Asn Pro Pro Asp His Ser Ala Pro Leu Gl - #y Val Thr Arg Pro Ser         #                15                                                           - Ala Pro Pro Leu Ala                                                                     20                                                                - (2) INFORMATION FOR SEQ ID NO:2:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 19 amino                                                          (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                 - Pro Ser Ala Pro Pro Leu Pro His Val Val As - #p Leu Pro Gln Leu Gly         #                15                                                           - Pro Arg Arg                                                                 - (2) INFORMATION FOR SEQ ID NO:3:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 10 amino                                                          (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                 - Cys Pro Arg His Arg Pro Val Ser Arg Leu                                     #                10                                                           - (2) INFORMATION FOR SEQ ID NO:4:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 15 amino                                                          (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                 - Ser Pro Ser Gln Ser Pro Ile Phe Ile Gln Pr - #o Thr Pro Ser Gly             #                15                                                           - (2) INFORMATION FOR SEQ ID NO:5:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 22 amino                                                          (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                 - Arg Val Ser Arg Tyr Ser Ser Thr Ala Arg Hi - #s Arg Leu Arg Arg Gly         #                15                                                           - Ala Asp Gly Thr Ala Glu                                                                 20                                                                - (2) INFORMATION FOR SEQ ID NO:6:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 16 amino                                                          (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                 - Asp Lys Gly Ile Ala Ile Pro His Asp Ile As - #p Leu Gly Glu Ser Arg         #                15                                                           - (2) INFORMATION FOR SEQ ID NO:7:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 19 amino                                                          (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                                 - Asp Tyr Asp Asn Gln His Glu Gln Asp Arg Pr - #o Thr Pro Ser Pro Ala         #                15                                                           - Pro Ser Arg                                                                 - (2) INFORMATION FOR SEQ ID NO:8:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 18 amino                                                          (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                                 - Arg Pro Leu Gly Leu Gln Gly Cys Ala Phe Gl - #n Ser Thr Val Ala Glu         #                15                                                           - Leu Gln                                                                     - (2) INFORMATION FOR SEQ ID NO:9:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 20 amino                                                          (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                                 - Gln Ser Thr Val Ala Glu Leu Gln Arg Leu Ly - #s Met Lys Val Gly Lys         #                15                                                           - Thr Arg Glu Leu                                                                         20                                                                - (2) INFORMATION FOR SEQ ID NO:10:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 21 amino                                                          (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:                                - Ala Asn Gln Pro Gly His Leu Ala Pro Leu Gl - #y Glu Ile Arg Pro Ser         #                15                                                           - Ala Pro Pro Leu Ala                                                                     20                                                                - (2) INFORMATION FOR SEQ ID NO:11:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 19 amino                                                          (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:                                - Pro Ser Ala Pro Pro Leu Pro Pro Val Ala As - #p Leu Pro Gln Pro Gly         #                15                                                           - Leu Arg Arg                                                                 - (2) INFORMATION FOR SEQ ID NO:12:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 20 amino                                                          (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:                                - Thr Ser Val Glu Asn Ala Gln Gln Asp Lys Gl - #y Ile Ala Ile Pro His         #                15                                                           - Asp Ile Asp Leu                                                                         20                                                                - (2) INFORMATION FOR SEQ ID NO:13:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 19 amino                                                          (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:                                - Asn Thr Thr Ala Ser Asp Gln Leu Leu Val Gl - #u Asn Ala Ala Gly His         #                15                                                           - Arg Val Ala                                                                 - (2) INFORMATION FOR SEQ ID NO:14:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 12 amino                                                          (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:                                - Pro Tyr Ile His Pro Thr Asn Pro Phe Ala Pr - #o Asp                         #                10                                                           - (2) INFORMATION FOR SEQ ID NO:15:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 18 amino                                                          (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:                                - Gly Ser Ala Trp Arg Asp Gln Ala Gln Arg Pr - #o Ala Val Ala Ser Arg         #                15                                                           - Arg Arg                                                                     - (2) INFORMATION FOR SEQ ID NO:16:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 14 amino                                                          (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:                                - Glu Tyr Asp Gln Ser Thr Tyr Gly Ser Ser Th - #r Gly Pro Val                 #                10                                                           - (2) INFORMATION FOR SEQ ID NO:17:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 14 amino                                                          (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:                                - Leu Asp Gly Arg Pro Leu Ser Thr Ile Gln Gl - #n Tyr Ser Lys                 #                10                                                           - (2) INFORMATION FOR SEQ ID NO:18:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 23 amino                                                          (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:                                - Asp Thr Leu Asp Tyr Pro Ala Arg Ala His Th - #r Phe Asp Asp Phe Cys         #                15                                                           - Pro Glu Cys Arg Pro Leu Gly                                                             20                                                                - (2) INFORMATION FOR SEQ ID NO:19:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 15 amino                                                          (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:                                - Pro Thr Pro Ser Pro Pro Met Ser Pro Leu Ar - #g Pro Gly Leu Asp             #                15                                                           - (2) INFORMATION FOR SEQ ID NO:20:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 18 amino                                                          (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:                                - Thr Pro Gly Asn Thr Asn Thr Arg Val Ser Ar - #g Tyr Ser Ser Thr Ala         #                15                                                           - Arg His                                                                     __________________________________________________________________________

What is claimed is:
 1. A method of detecting present or previoushepatitis E viral infection in a subject, comprising the steps of:a.contacting an antibody-containing sample from the subject with adetectable amount of an antigenic composition comprising at least fourdifferent peptides, wherein the peptides have a sequence consisting ofthe amino acids contained in the amino acid sequences defined in theSequence Listing by SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:8 and SEQ IDNO:9 to form an antigen-antibody complex; b. detecting theantigen-antibody complex, the presence of the complex indicating thepresence of present or previous hepatitis E infection.
 2. The method ofclaim 1, wherein the sample comprises serum.
 3. A method of detectingpresent or previous hepatitis E viral infection in a subject comprisingthe steps of:a. contacting an antibody-containing sample from thesubject with a detectable amount of an antigenic composition comprisingat least five different peptides, wherein the peptides have a sequenceconsisting of the amino acids contained in the amino acid sequencesdefined in the Sequence Listing by SEQ ID NO:1, SEQ ID NO:2, SEQ IDNO:7, SEQ ID NO:8 and SEQ ID NO:9 to form an antigen-antibody complex;b. detecting the antigen-antibody complex, the presence of the complexindicating the presence of present or previous hepatitis E infection. 4.The method of claim 3, wherein the sample comprises serum.
 5. A methodof detecting present or previous hepatitis E viral infection in asubject comprising the steps of:a. contacting an antibody-containingsample from the subject with a detectable amount of an antigeniccomposition comprising antigenically effective amounts of at least eightdifferent peptides, wherein the peptides have a sequence consisting ofthe amino acids contained in the amino acid sequences defined in theSequence Listing by SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:6,SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10 and SEQ ID NO:11 to form anantigen antibody complex; b. detecting the antigen-antibody complex, thepresence of the complex indicating the presence of present or previoushepatitis E infection.
 6. The method of claim 3, wherein the samplecomprises serum.
 7. A method for diagnosing the acute phase of hepatitisE infection in a subject, comprising the steps of:a. contacting anantibody-containing sample from the subject with a detectable amount ofan antigenic composition comprising antigenically effective amounts ofat least two different peptides, wherein the peptides have a sequenceconsisting of the amino acids contained in the amino acid sequencesdefined in the Sequence Listing by SEQ ID NO:1 and SEQ ID NO:2 to forman antigen-antibody complex; b. detecting the presence of the complex,the presence of the complex indicating the presence of the acute phaseof hepatitis E infection.
 8. The method of claim 7, wherein the samplecomprises serum.